An ongoing NCI trial has underscored the capacity of adoptively transferred T cells to achieve clinically meaningful responses in melanoma patients. This project employs clinically predictive mouse models of adoptive immunotherapy (AIT) to obtain mechanistic and translational guidance, which would be difficult to obtain solely through direct clinical observations. Our laboratory recently identified that a small L-selectin low subset of T cells present in tumor-draining lymph nodes includes naturally sensitized antitumor CD8 + effector T cells (TE) with helper-independent function. Helper-independence is defined by the observed ability of these CD8 + TE to cure established tumors when they are administered in sufficient "stand alone" doses, without co-administration of CD4 + TE or adjunct treatments such as rIL-2. Our aims focus on three key elements of successful AIT: (1) TE culture conditions can dictate therapeutic outcome in the absence of apparent avidity or affinity selection forces; (2) failure to purge tumor-induced suppressor T cells (Ts) during culture can subvert the effector phase of AIT; (3) although administration of adjuncts such as IL-2 remains unnecessary, cure of extrapulmonary tumors continues to require adjunct chemotherapy or radiation therapy (RT). In regards to Component 1, we are identifying culture modifications which not only hyperexpand L-selectin lxw TE, but which also strikingly enhance their therapeutic potency. These modifications do not increase T cell avidity or detectably modulate T-cell receptor (TCR) repertoires, but do lead to a markedly enhanced capacity of L-selectin low TE to withstand apoptosis at TCR reengagement. Such modifications have furthermore enabled recovery of fully potent Lselectin low TE even from pre-terminal mice. We are characterizing the mechanism(s) of enhanced TE resistance to apoptosis and its relation to enhanced therapeutic potency. In regards to Component 2, we are investigating the mechanism(s) by which passenger Ts exert effector blockade, with the goal of delineating phenotypic or functional distinctions to enable their quantitative removal prior to adoptive transfer. In regards to Component 3, we have identified that sublethal irradiation results in peripheral blood mobilization of CD34 + cells with up-regulated co-stimulatory molecule expression. We are characterizing the capacity of such cells to enhance intratumoral antigen presentation, and to facilitate successful AIT in hosts, which have not received RT or chemotherapy.